(A) Study pathogenesis and pathophysiology of high biocontainment viral pathogens utilizing molecular technologies including reverse genetics systems: Filoviruses: The Syrian hamster model for Ebola virus infection displays almost all hallmark features of human disease including coagulopathy. Using this model, we have investigated the mechanisms in which hamsters are protected from wild-type Ebola virus infection and determined that a CD4+ T cell response, facilitating a neutralizing antibody response, is responsible for natural immunity. Ebola virus uses transcriptional editing to express several glycoproteins from a single gene. Recombinant viruses predominantly expressing the transmembrane glycoprotein are known to rapidly mutate and acquire an editing site predominantly expressing a soluble form of the glycoprotein, suggesting an important role of this protein. Using reverse genetics, however, we could show that the soluble glycoprotein is not required for virulence in the guinea pig model. Utilizing mice from the Collaborative Cross (CC), a panel of reproducible, recombinant inbred strains spanning the genetic breadth of three murine subspecies, we could demonstrate variation in susceptibility to distinct disease phenotypes following Ebola virus infection. In contrast to C57BL/6J mice, which develop lethal disease without coagulopathies, CC recombinant inbred intercross (CC-RIX) lines develop either complete resistance to lethal disease or lethal disease characterized by prolonged coagulation times. Transcriptomics revealed that the host responses dependent on genetic background determine susceptibility to infection independent of virus replication. (B) Study immune responses to infection and vaccination of high containment viral pathogens and develop new vaccine candidates: Arenaviruses: Vaccine development has continued with the vesicular stomatitis virus (VSV) platform expressing the Josiah strain glycoprotein. Depletion studies in the cynomolgus macaque model indicated the importance of cellular and humoral immunity for protection mediated by the VSV based vaccine. Furthermore, we could demonstrate protection against challenge with geographically and genetically distinct Lassa viruses. Thus, the VSV based vaccine platform can be considered a pan-Lassa vaccine approach and GMP production has been initiated. In addition, we have started a project focused on the elimination of Lassa virus from the reservoir species, Mastomys natalensis. For this we use two vaccine platforms, a recombinant attenuated strain of Salmonella and a recombinant cytomegalovirus vector, to immunize the peri-domestic reservoir with the hope to block Lassa virus transmission among rodents and to humans. Bunyaviruses: We have continued to characterize and optimize the adenovirus based vaccine for Crimean-Congo hemorrhagic fever virus (CCHF). Ongoing transfer and depletion studies using the adenovirus-based vaccines expressing the nucleoprotein or glycoproteins of CCHF virus indicated that antibodies are the mechanism of protection. We also developed a VSV-based vaccine expressing the CCHF glycoprotein. Characterization and efficacy testing are ongoing. Filoviruses: The VSV vaccine efforts are reported under the 'Trivalent Filovirus Vaccine' project. In the meantime, we have proceeded with the development of alternative vaccine candidates for Ebola and Marburg viruses. One of the approaches is based on a whole-virus vaccine using an Ebola virus lacking an indispensable gene encoding for the virion protein VP30. This vaccine showed promising protection in the macaque model following a single administration. Protection was also achieved after prime-boost immunization with an inactivated form of the whole-virus vaccine. Another approach is targeted towards wildlife, in particular the great apes, using the concept of a disseminating vaccine vector. The idea is the introduction of a recombinant CMV expressing the Ebola virus glycoprotein. Proof-of-concept studies have been successful in the mouse and rhesus macaque model using a murine and rhesus CMV vector, respectively. Very recently, we have started to investigate a vaccine based on Modified Vaccinia Ankara (MVA) expressing filovirus-like particles; preliminary rodent experiments have resulted in promising results. (C) Study vector/reservoir transmission of high containment viral pathogens using appropriate animal models: Arenaviruses: Over the past months we have established a Mastomys natalensis colony at Rocky Mountain Laboratories using animals imported from Mali. This unique resource will allow us to study infection and transmission in the natural reservoir species. The colony is susceptible to infection which different Lassa strains and infection leads to temporal virus persistence followed by clearance. More recent studies include temporal analysis of Lassa-Soromba infection in adult Mastomys as well as vertical and horizontal transmission among animals. (D) Utilize in vitro and in vivo systems to study the interactions between viral pathogen or viral components and host cells and develop new antiviral strategies: Arenaviruses: We have tested the efficacy of two antivirals, T-705 and ribavirin, in the guinea pig model of Lassa virus infection. In particular, T-705 showed promise as a treatment for Lassa virus with protection even when treatment was started post-disease onset (daily dose of 300mg/kg). Studies testing the efficacy of T-705 against Lujo virus, a recently emerged arenavirus, will begin shortly. Filoviruses: Ribavirin can effectively extend the time-to-death of hamsters infected with Ebola virus, but resistance will rapidly develop. Surprisingly, T-705 protected hamsters against Ebola virus infection when animals were treated for two weeks beginning the day after infection. This promising treatment option is currently being followed up. We have also identified a few promising broad spectrum antivirals against Ebola virus, which are in development as treatment options for other RNA viruses. Further confirmation is underway. In contrast, treatment with anti-malaria drugs, including chloroquine, failed in Ebola rodent models and should not be further considered. We have continued to develop novel experimental systems that allow modeling of the filo- and arenavirus life cycle over multiple infectious cycles without the need for a high containment laboratory. We have started to utilize those systems for drug screening experiments to identify viral and host factors involved in virus replication. Preliminary data have identified several host factors and we are in the process to confirm their contribution to virus replication. These systems will be extremely helpful for future drug development efforts. (E) Study the epidemiology and ecology of high biocontainment pathogens utilizing newly developed rapid, sensitive and specific diagnostic test systems including those that can be applied under field conditions: Arenaviruses: Studies regarding the ecology and epidemiology of Lassa virus are reported under the Mali ICER project. Filoviruses: Over the past fiscal year we have established and operated a diagnostic laboratory in Monrovia, Liberia to support the international response to the ongoing Ebola outbreak in West Africa. For this, we have established molecular and serological detection assays that were transferred from our ICER field sites. In addition, we established and supported an Ebola diagnostic laboratory in Bamako, Mali. Overall, we have analyzed over 5000 specimens at both locations. Our work in West Africa has further contributed to a better understanding of the molecular epidemiology of Ebola. Most importantly, we have implemented a new sequence device, MinION, which allows full-genome determination on site under rural field conditions.